1. Field of the Invention
The present invention relates to a protein which induces the interferon-.gamma. (hereinafter abbreviated as "IFN-.gamma.") production by immunocompetent cells, and a monoclonal antibody specific to the protein.
2. Description of the Prior Art
It is said that IFN-.gamma. is a protein which has antiviral-, antioncotic- and immunoregulatory-activities and which is produced by immunocompetent cells stimulated with antigens and/or mitogens. Because of these biological activities, IFN-.gamma. is expected to be used as an antitumor agent since the discovery, and energetically studied on clinical trials as a therapeutic agent for malignant tumors in general including brain tumors. IFN-.gamma. preparations now commercially available are roughly classified into 2 groups, i.e. natural IFN-.gamma.s produced by immunocompetent cells and recombinant IFN-.gamma.s produced by transformants prepared by introducing DNAs which encode the natural IFN-.gamma.s into microorganisms of the species Escherichia coli. In such clinical trials, either of these IFN-.gamma.s is administered to patients as an "exogenous IFN-.gamma.".
Among these IFN-.gamma.s, the natural IFN-.gamma. is usually produced by culturing established immunocompetent cells in nutrient culture media supplemented with IFN-.gamma. inducers to form IFN-.gamma., and purifying the IFN-.gamma.. It is known that the type of IFN-.gamma. inducers greatly influence on the production yield and the facility of IFN-.gamma. purification, as well as the safeness of the final products. Generally, mitogens such as concanavalin A (Con A), Lens culinaris, Phytolacca americana, endotoxin and lipopolysaccharide are used. These mitogens, however, have problems of their molecular and quality varying dependently on their origins and purification methods, as well as the difficulty of obtaining a desired amount of preparations with a constant IFN-.gamma. inducibility. In addition, most of these mitogens induce unfavorable side effects when administered to living bodies, and some of them may cause toxicity, so that it is substantially difficult to induce the IFN-.gamma. production by the direct administration to living bodies.
During the study of cytokines produced by mammalian cells, the present inventors have found in mouse liver a novel substance which induces the IFN-.gamma. production. They isolated the substance using two or more conventional purification methods including column chromatography mainly, studied the property and feature and revealing that the reality is a protein having the following physicochemical properties:
(1) Molecular weight 19,000.+-.5,000 daltons on gel filtration sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE); PA1 (2) Isoelectric point (pI) 4.8.+-.1.0 on chromatofocusing; PA1 (3) Partial amino acid sequence Possessing partial amino acid sequences corresponding to amino acid residues 26-43 and 79-103 of SEQ ID NO:2 PA1 (4) Biological activity Inducing the interferon-.gamma. production by immunocompetent cells. PA1 (1) Molecular weight 19,000.+-.5,000 daltons on gel filtration and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE); PA1 (2) Isoelectric point (pI) 4.8.+-.1.0 on chromatofocusing; PA1 (3) Partial amino acid sequence Possessing partial amino acid sequences corresponding to amino acid residues 26-43 and 79-103 of SEQ ID NO:2 PA1 (4) Biological activity Inducing the IFN-.gamma. production by immunocompetent cells. PA1 (1) Molecular weight 19,000.+-.5,000 daltons on gel filtration and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE); PA1 (2) Isoelectric point (pI) 4.8.+-.1.0 on chromatofocusing; PA1 (3) Partial amino acid sequence Possessing partial amino acid sequences corresponding to amino acid residues 26-43 and 79-103 of SEQ. ID NO:2 PA1 (4) Biological activity Inducing the IFN-.gamma. production by immunocompetent cells.
Such a protein with these physicochemical properties has never been reported, and the data concludes that the protein is novel. The present inventors energetically studied on mouse liver cells and have succeeded to isolate a DNA encoding the protein. The decoding of the protein revealed that the DNA consists of 471 base pairs and encodes the amino acid sequence in SEQ ID NO:3. When the DNA was introduced into microorganisms of the species Escherichia coli to express the production of the present protein, the protein was produced in the culture in a satisfactorily high yield. These findings are disclosed in Japanese Patent Application No.184,162/94 applied by the present applicant.
As is described above, the present protein has an activity of inducing the IFN-.gamma. production by immunocompetent cells, and is expected to be used in a variety of uses as an anti-virus agent, antioncotic agent, antiseptic, immunoregulatory agent or a platelet-increasing agent. Generally, in the case of incorporating biologically active proteins into pharmaceuticals, the developments of methods for purifying such proteins highly and effectively and those for assaying samples containing these proteins are inevitable. The material most suitable for the purification and assay is a monoclonal antibody, but such a monoclonal antibody specific to the protein is not established.